Abstract A universal influenza vaccine that protects against all strains of influenza is a high priority. Seasonal influenza epidemics account for >200,000 hospitalizations and >30,000 deaths each year in the U.S., with more than 90% of the deaths occurring in the elderly. The Centers for Disease Control & Prevention (CDC) estimate the economic impact of seasonal influenza in the United States ranges from $10 to $16 billion and pandemic influenza ranges from $71.3 to $166.5 billion. The proposed research directly addresses the limitations of both pandemic and seasonal flu vaccines. Versatope licensed a unique technology platform that includes an influenza antigen construct that is potentially effective against several strains of influenza in mouse and ferret animal models. It is expressed in Escherichia coli and derived from recombinant outer membrane vesicles (rOMVs) that contains four repeats of diverse sequences from the ectodomain of the genetically conserved ion channel and drug target, M2 (4xM2e-rOMVs). The ferret study showed higher levels of antigen-specific antibodies and lower viral loads of a human pandemic H1N1 influenza isolate following prime/boost immunization of 4xM2e- rOMVs compared to a commercially available influenza vaccine. The rOMV vaccine delivery is innovative because our E. coli production strain has been genetically engineered to detoxify lipopolysaccharide (LPS) more than 1000-fold (and does not require chemical extraction of LPS to detoxify the final product) and produces rOMVs more than 30-fold compared to the parental probiotic strain of BSL1 bacteria. Since it is known that influenza undergoes antigenic variation under immunologic selection, the M2 ectodomain alone may not be a sustainable vaccine candidate for long-term preventative applications in humans. Although our current 4xM2e- rOMV vaccine candidate contains diverse influenza sequences from one target antigen, we propose to develop a multi-antigen influenza vaccine based on this candidate and to demonstrate improved protection against two different strains of influenza (H1N1 and H3N2) in the mouse model. We expect that our approach using multi- antigen (conserved domains from representative hemagglutinin, neuraminidase and nucleoprotein together with M2 ectodomains) influenza construct will yield stable rOMVs and provide protection against multiple influenza strains by focusing the immune response to genetically conserved domains representing influenza antigenic diversity. rOMVs are ideally suited for a multi-valent vaccine because the recombinant proteins can be expressed as fusion proteins and/or independently targeted to the lumen, the membrane, or the surface of rOMVs. Our proposed research program is primarily translational, the outcome of which will guide the path toward a viable single-dose vaccine for seasonal and pandemic influenza. The development of this new multivalent influenza- rOMV will enable large-scale production suitable for non-clinical development, toxicology, clinical studies, and commercial development. We will also identify the minimum dose required for immunogenicity for future safety/toxicity studies. These rOMVs represent a potentially safe and simple subunit vaccine delivery platform that will increase the range of protection against multiple strains of pandemic and seasonal influenza and reduce the overall hospitalization mortality and economic impact.